This project is directed toward characterizing the structure and function of tetanus toxin to understand how this toxin impairs neuronal cell function. The toxicity of tetanus toxin results from its proteolytic cleavage of the synaptic vesicle associated protein VAMP and subsequent inhibition of exocytosis. Both VAMP and its nonneuronal homologue, cellubrevin, appear to be important proteins in membrane trafficking and vesicle fusion events. To determine if tetanus toxin also interferes with exocytosis in nonneuronal cells (which lack receptors for the toxin), a fusion protein consisting of the enzymatically active domain of tetanus toxin and the receptor binding domain of anthrax toxin was prepared by standard recombinant DNA methodologies. This protein, LF-LC, inhibited neurotransmitter release similarly to tetanus toxin. LF-LC was cytotoxic to and inhibited transferrin receptor recycling in Chinese hamster ovary cells and several mouse macrophage cell lines, but was not cytotoxic in other cell lines including RBL-2H3 and Vero cells. Based upon mutagenesis studies, the cytotoxic effect of LF-LC appears to be due to its ability to cleave cellubrevin. These data suggest that cellubrevin is essential for growth in certain cell lines. LF-LC is being used to study the function of cellubrevin and for elucidating different vesicle trafficking pathways. In addition, LF-LC will be important in studying tetanus toxin activity in neuronal cells.Publications:Bartels, F., Bergel, H., Bigalke, H., Frevert, J., Halpern, J., and Middlebrook, J. (1994) Specific antibodies against the zinc-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin. J. Biol. Chem. 269:8122-8127.Arora, N., Williamson, L.C., Leppla, S.H., and Halpern, J.L. (1994) Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non- neuronal cells. J. Biol. Chem. (in press).